Altered monocyte cyclo‐oxygenase response in non‐obese diabetic mice

Monocytes infiltrate islets in non‐obese diabetic (NOD) mice. Activated monocyte/macrophages express cyclo‐oxygenase‐2 (COX‐2) promoting prostaglandin‐E₂ (PGE₂) secretion, while COX‐1 expression is constitutive. We investigated in female NOD mice: (i) natural history of monocyte COX expression basally and following lipopolysaccharide (LPS) stimulation; (ii) impact of COX‐2 specific inhibitor (Vioxx) on PGE₂, insulitis and diabetes. CD11b⁺ monocytes were analysed for COX mRNA expression from NOD (n = 48) and C57BL/6 control (n = 18) mice. NOD mice were treated with either Vioxx (total dose 80mg/kg) (n = 29) or methylcellulose as control (n = 29) administered by gavage at 4 weeks until diabetes developed or age 30 weeks. In all groups, basal monocyte COX mRNA and PGE₂ secretion were normal, while following LPS, after 5 weeks of age monocyte/macrophage COX‐1 mRNA decreased (P < 0·01) and COX‐2 mRNA increased (P < 0·01). However, diabetic NOD mice had reduced COX mRNA response (P = 0·03). Vioxx administration influenced neither PGE₂, insulitis nor diabetes. We demonstrate an isoform switch in monocyte/macrophage COX mRNA expression following LPS, which is altered in diabetic NOD mice as in human diabetes. However, Vioxx failed to affect insulitis or diabetes. We conclude that monocyte responses are altered in diabetic NOD mice but COX‐2 expression is unlikely to be critical to disease risk.     

IL‐1β/HMGB1 Complexes Promote The PGE2 Biosynthesis Pathway in Synovial Fibroblasts

PGE₂ is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase‐1 (mPGES‐1) is a terminal enzyme of the PGE₂ pathway inducible by proinflammatory cytokines. mPGES‐1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High‐mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro‐arthritogenic role in RA. We aimed to investigate the effects of IL‐1β/HMGB1 complexes on mPGES‐1 and other enzymes of the PGE₂ pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX‐2 inhibition and IL‐1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL‐1β significantly increased mPGES‐1 and COX‐2 expressions as well as PGE₂ production, as compared to treatment with HMGB1 or IL‐1β alone. Furthermore, NS‐398 reduced the production of IL‐6 and IL‐8, thus indicating that IL‐1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL‐1RA completely abolished the induced PGE₂ and cytokine production, suggesting an effect mediated through IL‐1RI. IL‐1β/HMGB1 complexes promote the induction of mPGES‐1, COX‐2 and PGE₂ in SF. The amplification of the PGE₂ biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.     

Dual inhibition of 5-lipoxygenase/cyclooxygenase by a reconstituted homeopathic remedy; possible explanation for clinical efficacy and favourable gastrointestinal tolerability

Objective: In order to elucidate potential anti-inflammatory activities of Zeel comp. N and its constituents, the inhibition of the synthesis of Leukotriene B₄ (LTB₄) and Prostaglandin (PGE₂) by 5-lipoxygenase (5-LOX) and cyclo-oxygenase 1 and 2 (COX 1 and 2) respectively were examined in vitro.Materials: Human HL-60 cells, differentiated for 6–8 days with DMSO (1.2% v/v) were used for the 5-LOX assay. The COX activity assays were carried out with purified enzymes, COX 1 (ram seminal vesicles), COX 2 (sheep placenta) and with human THP-1 cells, differentiated for 24 h with PMA (50 nM).Methods: LTB₄ and PGE₂ production in the 5-LOX and COX assays respectively were determined by enzyme linked immunoassays.Results: A reconstituted Zeel comp. N combination as well as its constituent mother tinctures of Arnica montana, Sanguinaria canadensis and Rhus toxicodendron (Toxicodendron quercifolium) showed distinct inhibitory effects on the production of LTB₄ by 5-LOX (IC₅₀ values of 10, 20, 2 and 5 µg/ml respectively) and on the synthesis of PGE₂ by COX 1 (IC₅₀ values of 50, 80, 40 and 20 µg/ml respectively) and COX 2 enzymes (IC₅₀ values of 60, 110, 50 and 20 µg/ml respectively). The mother tincture of Solanum dulcamara inhibited the production of PGE₂ by COX 1 (IC₅₀ 40 µg/ml) and COX 2 (IC₅₀ 150 µg/ml) but not production of leukotriene LTB₄ by 5-LOX.Conclusions: The observed dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and the favorable gastrointestinal tolerability of the original remedy Zeel comp. N.Received 10 April 2003; returned for revision 27 May 2003; accepted by W. B. van den Berg 24 November 2003     

COX‐2‐Mediated Regulation of VEGF‐C in Association With Lymphangiogenesis and Lymph Node Metastasis in Lung Cancer

The mechanisms underlying the effects of COX‐2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83‐a with different metastatic capacities were investigated by immunostaining, western blotting, and real‐time RT‐PCR. We observed increased expressions of COX‐2 and VEGF‐C in the three highly metastatic cell lines compared with the less metastatic AGZY83‐a cell line. The COX‐2‐specific inhibitor Celecoxib suppressed VEGF‐C expression whereas the main COX‐2 metabolite PGE₂ elevated VEGF‐C expression in Anip973 and AGZY83‐a cells in positive and negative experiments. To determine the functional link to COX‐2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX‐2 expression in Anip973 cells and transfected a COX‐2 cDNA to enhance the low COX‐2 expression in AGZY83‐a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF‐C production in the COX‐2‐knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF‐C production in the COX‐2‐overexpressing AGZY83‐a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF‐C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE₂ treatment increased the same factors in the parental AGZY83‐a cells. A positive correlation between COX‐2 and VEGF‐C was also confirmed in vivo. The present data suggest that COX‐2 regulates VEGF‐C expression via the PGE₂ pathway, and that EP1/EP4 receptors are involved in PGE₂‐mediated VEGF‐C production. Thus, COX‐2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The fish oil ingredient,docosahexaenoic acid,activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A₂ (cPLA₂) and lipid mediators produced from cPLA₂ activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA₂ and cyclooxygenase 2 (COX‐2), and cause prostaglandin E₂ (PGE₂) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA₂ via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA₂ activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA₂, COX‐2 and production of PGE₂ as a cPLA₂ inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA₂ product arachidonic acid and PGE₂ also play an anti‐inflammatory role. This effect of PGE₂ is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE₂ because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE₂.     

Inflammatory mediator changes in cotton-top tamarins (CTT) after SC-41930 anti-colitic therapy

Use of the CTT model provides insight into the inflammatory mediator contribution in the pathogenesis of idiopathic colitis. To evaluate anti-colitic efficacy, the leukotriene B₄ receptor antagonist and anti-inflammatory agent, SC-41930, was administered (10 mg/kg BW by gavage BID) for 8 weeks to CTTs with histologically confirmed persistent and defined active colitis. The inflammatory mediators LTB₄, PGE₂, TXB₂, and PAF were assayed in colonic dialysate that was collected after 11/2 h from four CTTs pre-, mid-, and post-treatment, frozen at −70°C, and analyzed by RIA after HPLC purification. LTB₄ levels were lower at mid- and post-treatment and had little inter-animal variation post-treatment. PGE₂ and PAF levels were elevated during SC-41930 treatment, but there was a trend towards lower thromboxane B₂ levels. Reduced LTB₄ (PMN degranulation and chemotaxis) and increased PGE₂ (mucosal-protective effect) may, in part, explain the observed efficacy of SC-41930 in active tamarin colitis.

     

Effects of celecoxib on major prostaglandins in asthma

Background Prostaglandin (PG) D₂ is a pro‐inflammatory and bronchoconstrictive mediator released from mast cells, and is currently evaluated as a new target for treatment of asthma and rhinitis. It is not known which cyclooxygenase (COX) isoenzyme catalyses its biosynthesis in subjects with asthma. Objectives Primarily, to assess whether treatment with the COX‐2 selective inhibitor celecoxib inhibited biosynthesis of PGD₂, monitored as urinary excretion of its major tetranor metabolite (PGDM). Secondarily, to determine the effects of the treatment on biosynthesis of PGE₂, thromboxane A₂ and PGI₂, also measured as major urinary metabolites. Methods Eighteen subjects with asthma participated in a cross‐over study where celecoxib 200 mg or placebo were given b.i.d. on 3 consecutive days following 2 untreated baseline days. Six healthy controls received active treatment with the same protocol. Urinary excretion of the eicosanoid metabolites was determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Lung function was followed as FEV₁ and airway inflammation as fraction of exhaled nitric oxide (F_ENO). Results Celecoxib treatment inhibited urinary excretion of PGEM by 50% or more in subjects with asthma and healthy controls, whereas there was no significant change in the excretion of PGDM. In comparison with the healthy controls, the subjects with asthma had higher baseline levels of urinary PGDM but not of PGEM. The 3‐day treatment did not cause significant changes in FEV₁ or F_ENO. Conclusion and Clinical Relevance Biosynthesis of PGD₂ was increased in subjects with asthma and its formation is catalysed predominantly by COX‐1. By contrast, COX‐2 contributes substantially to the biosynthesis of PGE₂. The asymmetric impact of celecoxib on prostanoid formation raises the possibility of long‐term adverse consequences of COX‐2 inhibition on airway homeostasis by the decreased formation of bronchodilator PGs and maintained production of increased levels of bronchoconstrictor PGs in asthmatics. Cite this as: K. Daham, W.‐ L. Song, J. A. Lawson, M. Kupczyk, A. Gülich, S.‐E. Dahlén, G. A. FitzGerald and B. Dahlén, Clinical & Experimental Allergy, 2011 (41) 36–45.     

Calcium/calmodulin‐dependent protein kinase II regulates cyclooxygenase‐2 expression and prostaglandin E2 production by activating cAMP‐response element‐binding protein in rat peritoneal macrophages

Prostaglandin E₂ (PGE₂) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE₂ production is regulated by arachidonic acid release and cyclooxygenase‐2 (COX‐2) expression. In the present study, we found that COX‐2 expression can be achieved by activating Ca²⁺/Calmodulin (CaM)‐dependent protein kinase II (CaMKII) and cAMP‐response element‐binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca²⁺]_i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX‐2 expression and PGE₂ production in a chronological order. These results suggested that Ca²⁺/CaM‐dependent CaMKII plays an important role in mediating COX‐2 expression and PGE₂ production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE₂ production, which plays an essential role in inflammation and cancers.     

Triggering of DC migration by dengue virus stimulation of COX‐2‐dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E₂ (PGE₂) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE₂ pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE₂ can induce CCR7 expression on DC and that antagonists of the PGE₂ receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE₂ on the migration of DV‐infected DC.     

Effects of topical corticosteroids on inflammatory mediator–induced eicosanoid release by human airway epithelial cells

Background: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. Objectives: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. Methods: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 μmol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE₂, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA₂], and Clara cell protein, a PLA₂ inhibitor) was determined by Western blotting. Results: Eicosanoid output was largely confined to prostaglandins with values of 5 ± 2 and 82 ± 35 ng PGE₂/10⁶ cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE₂ output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE₂ output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE₂ inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA₂ (Type IV), or iPLA₂ (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. Conclusion: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis. (J Allergy Clin Immunol 1999;103:1081-91.)     

A regulatory cross‐talk between Vγ9Vδ2 T lymphocytes and mesenchymal stem cells

The physiological functions of human TCRVγ9Vδ2⁺ γδ lymphocytes reactive to non‐peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVγ9Vδ2⁺ lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B‐cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of γδ‐cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX‐2‐dependent production of prostaglandin E2 (PGE₂) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vγ9Vδ2 T lymphocytes. COX‐2 expression and PGE₂ production by MSC were not constitutive, but were induced by IFN‐γ and TNF‐α secreted by activated Vγ9Vδ2 T cells. This regulatory cross‐talk between MSC and Vγ9Vδ2 T lymphocytes involving PGE₂ could be of importance for the antitumor and antimicrobial activities of γδ T cells.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.     

Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.

     

M2000 (β‐D‐Mannuronic Acid) as a Novel Antagonist for Blocking the TLR2 and TLR4 Downstream Signalling Pathway

To date, selective blockade of Toll‐like receptor (TLR) signalling has been developed as a new approach for treatment for many inflammatory diseases. As β‐D‐mannuronic acid (M2000) has been known as an anti‐inflammatory molecule in several experimental models, we investigated the antagonistic effects of M2000 on TLR2 and TLR4 downstream signalling transduction pathway in human embryonic kidney (HEK) 293 cell lines overexpressing TLR2/CD14 and the TLR4/MD2/CD14 complex, respectively. M2000 effectively inhibited mRNA expression of MyD88 and p65, major subunit of nuclear factor‐κB, in HEK293 cells stimulated by lipoteichoic acid (LTA, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) with no evidence of cytotoxicity. In addition, M2000 also suppressed LTA and LPS‐induced production of TNF‐α and IL‐6 inflammatory cytokines in these cells. Furthermore, the results revealed that M2000 had no significant effect on Tollip mRNA expression as a negative regulator of TLR signalling in aforesaid cells. Overall, these data point to M2000 inhibitory effect on Toll‐like receptor (TLR) 2, 4 signalling in HEK293 cells. This information might provide new insights into the possible roles of this small drug in order to introduce it as a TLR signalling pathway inhibitor. However, more studies are needed to confirm β‐D‐mannuronic acid antagonistic effects including the effects of M2000 on peritoneal isolated macrophages and also on blood cells in patients with inflammatory diseases such as ankylosing spondylitis.     

Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2‐induced neutrophil migration by regulating IL‐8 secretion

Extracellular nucleotides regulate a variety of cellular responses involved in inflammation via the activation of P2 receptors. Here, we show that nucleotides regulate TLR2‐induced neutrophil migration both in vivo and in vitro. The nucleotide scavenger apyrase inhibited neutrophil recruitment in murine air pouches injected with the TLR2 agonist Pam₃CSK₄. In agreement, the supernatants of either human primary monocytes or monocytic cells (THP‐1 and U937) treated with Pam₃CSK₄ recruited significantly fewer neutrophils when the former cells were treated in the presence of apyrase. As demonstrated with inhibitory Ab, these supernatants induced neutrophil migration due to IL‐8 secretion. In addition, IL‐8 secretion was markedly diminished by the non‐selective P2 receptor antagonists reactive blue 2 and suramin, and by a selective P2Y₆ antagonist, MRS2578. Selective antagonists of P2Y₁ (MRS2500) and P2Y₁₁ (NF157) did not affect IL‐8 release. The knockdown of either P2Y₂ or P2Y₆ with specific shRNA diminished IL‐8 secretion from Pam₃CSK₄‐treated THP‐1 cells. Altogether, these results show that extracellular nucleotides, via P2Y₂ and P2Y₆ receptors, regulate neutrophil migration by controlling TLR2‐induced IL‐8 release from human monocytes. In line with our previous work on TLR4, this study further supports the importance of nucleotides in bacterial‐induced neutrophil migration.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 μM). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.

     

Dengue virus up‐regulates expression of notch ligands Dll1 and Dll4 through interferon‐β signalling pathway

The Notch signalling pathway is involved in multiple cellular processes and has been recently indicated to modulate the host immune response. However, the role of the Notch pathway in dengue virus (DENV) infection remains unknown. Our study has screened the expression profile of Notch receptors, ligands and target genes in human monocytes, macrophages and dendritic cells in response to DENV infection. The real‐time PCR data showed that Notch ligand Dll1 was significantly induced in DENV‐infected monocytes; and receptor Notch4, ligands Dll1 and Dll4, and target Hes1 were dramatically enhanced in DENV‐infected macrophages and dendritic cells. In macrophages, induction of Dll1 and Dll4 mediated by DENV2 was increased by treatment with interferon‐β (IFN‐β), and was impaired by neutralization of IFN‐β. The DENV‐induced Dll1 and Dll4 expression level was decreased by silencing key innate immune molecules, including Toll‐like receptor 3 (TLR3), MyD88, RIG‐I and IPS‐I. In IFN‐receptor‐depleted macrophages, the Dll1 and Dll4 induction was significantly alleviated. Functionally, activation of Notch signalling by Dll1 in CD4⁺ T cells enhanced the expression of a T helper type 1 (Th1) cytokine IFN‐γ, while Notch activation in macrophages had no direct effect on replication of DENV. Our data revealed that the expressions of Notch ligands in antigen‐presenting cells were differentially induced by DENV via innate immune signalling, which is important for Th1/Th2 differentiation during adaptive immune response.